Since the enzymes in eicosanoid oxidative cascades exclusively utilize free (i.e., non-esterified) arachidonic acid and the cellular content of non-esterified arachidonic acid in resting cells is exceedingly low (See: Samuelsson, B., et al. (1978) Annu. Rev. Biochem. 47:997-1029; Bills, T. K., et al. (1977) J. Clin. Invest. 60:1-6; Lands, W. E. M. (1984) Prostaglandins Leukotrienes Med. 13:35-46; Hemler M. E. and W. E. M. Lands (1980) J. Biol. Chem. 255:6253-61; Mizxuno, K., et al. (1982) Prostaglandins 23:743-57; and Needleman, P., et al. (1986) Annu. Rev. Biochem. 55:69-102), it seems evident that the activation of phospholipases A.sub.2 represents an enabling event in the generation of eicosanoid-based lipid second messengers. The release of arachidonic acid and the concomitant accumulation of amphiphilic reaction products modulate cellular responses to a wide variety of physiologic and pathophysiologic perturbations. The precise complement of downstream enzymes which determine the metabolic fate of released arachidonic acid is modulated by each cell's genetic program and by alterations in the physical and chemical environment of the activated cell. Thus, dynamic alterations in intracellular phospholipase A.sub.2 activity represent the critical step in the initiation of eicosanoid-based signaling cascades while the chemical interpretation of the signal reflects both the distant and proximal history of the activated cell.
Despite the importance of alterations in the activity of intracellular phospholipases in both physiologic and pathophysiologic processes, the precise identification of the biochemical mechanisms which regulate the activity of these enzymes has remained elusive. Although initial studies on the regulation of phospholipases A.sub.2 have focused in large part on the direct role of calcium ion in the activation of phospholipases A.sub.2 (See: Verheij, N. M. , et al., (1981) Rev. Physiol. Biochem. Pharmacol. 91: 91-203; van den Bosch, H. (1980) Biochim. Biophys. Acta. 604: 191-246; Dennis, E. A. (1983) Enzymes 3rd Ed. 307-353; Dennis, E. A. (1983) Prostaglandins, In the Enzymes P. D. Boyer, ed. (Academic: New York) 16:307-354; and Loeb, L. A. and R. W. Gross (1986) J. Biol. Chem. 261:10467-10470), it has recently become evident that additional biochemical mechanisms play essential roles in the regulation of intracellular phospholipases A.sub.2 (See: Zupan, L. A., et al., (1991) FEBS 284:27-30; Ghomashchi, F., et al. ( 1992) Biochem 31:3814-3824; Berg, O. G., et al. (1991) Biochem. 30:7283-7297; Jain, M. K., et al. (1991) Biochem. 30:7306-7317; and Jain, M. K., et al. (1991) Biochem. 30:7306-7340) . This was perhaps best exemplified by the identification of calcium-independent phospholipases A.sub.2 in a variety of cell types which manifest full catalytic activity and substrate affinity in the absence of calcium ion (.See: Wolf, R. A. and R. W. Gross (1985) J. Biol. Chem. 260:7295-7303; Hazen, S. L., et al., (1990) J. Biol. Chem. 265:10622-10630; Ross, M. I., et al., (1985) Arch. Biochem. Biophys. 238:247-258; Nijssen, J. G. et al., (1986) Biochim. Biophys. Acta 876:611-618; Husebye, E. S., and T. Flatmark (1987) Biochim. Biophys. Acta 920:120-130; Pierik, A. J., et al., (1988) Biochim. Biophys. Acta 962:345-353; and Gassama-Diagne A., et al., (1989) J. Biol. Chem. 264:9470-9475).
The recent demonstration that alterations in ATP concentration influence the activity and longevity of myocardial cytosolic calcium-independent phospholipase A.sub.2 and that myocardial cytosolic phospholipase A.sub.2 exists as a high molecular weight catalytic complex comprised of catalytic and regulatory polypeptides have provided initial insight into the importance of ligand modulated protein-protein interactions in the regulation of this class of enzymes.
Myocardial cytosolic calcium-independent phospholipase A.sub.2 has been reported to exist as a 400 kDa cytosolic complex that exhibits MCPA.sub.2 activity and can be purified from rabbit myocardial muscle tissue. In addition, MCPA.sub.2 activity was observed in the purified 40 kDa catalytic subunit of MCPA.sub.2 which has been shown to be active in a calcium-independent/ATP regulated manner in vitro (Hazen, S. L., and R. W. Gross (1991) J. Biol. Chem. 266:14526-14534).
The catalytic subunit of MCPA.sub.2 has been identified to be a 40 kDa protein which is considered unstable and is not regulated by ATP. Following the initial description (Wolf, R. A. and Gross, R. W. (1985) J. Biol. Chem. 260:7295-7303; Loeb, L. A. and Gross, R. W. (1986) J. Biol. Chem. 261:10467-10470), characterization (Kramer, R. M., et al. (1991) J. Biol. Chem 266:5268-5272; Leslie, C. C. , et al. (1988) Biochem. Biophys. Acta 963:476-492 and Hazen, S. L., et al. (1990) J. Biol. Chem. 265:10622-10630) and, in some cases, molecular cloning (Clark, J. D., et al (1991) Cell 65:1043-1051 and Zupan, L. A et al. (1992) J. Biol. Chem. 267:8707-8710) of intracellular phospholipases A.sub.2, attention has focused on the biochemical mechanisms responsible for their regulation.
Recent studies have demonstrated that myocardial cytosolic calcium-independent phospholipase A.sub.2 catalytic activity is regulated by protein-protein interactions which are modulated by ATP (Hazen, S. L. and Gross, R. W. (1991) J. Biol. Chem. 266:14526-14534). The 400 kDa complex that has been identified to contain MCPA.sub.2 activity is relatively stable and regulated by ATP.
Since activation of myocardial cytosolic calcium-independent phospholipase A.sub.2 has been implicated in the genesis of electrophysiologic dysfunction and myocytic cellular necrosis during myocardial ischemia Gross, R. W., (1992) Trends in Cardiovascular Medicine 2:115-121, the chemical identity of the elements responsible for the modulation of calcium-independent phospholipase A.sub.2 and the nature of the molecular mechanism through which ATP modulates the interactions between the catalytic and regulatory polypeptides have attracted considerable attention.
There is a need for a method of identifying compounds which modulate the activity of phospholipase A.sub.2. Such compounds would be useful to influence intracellular phospholipase activity involved in both physiologic and pathophysiologic processes and thereby affect those processes.